Buccutite™ Peroxidase (HRP) Antibody Conjugation Kit *Optimized for Labeling 25 ug Protein*

1. Add 1.25 µL Reaction Buffer (Component C) into antibody (25 µL).

2. Add 2.5 µL reconstituted Buccutite™ MTA(Component B).

3. Incubate at room temperature for 30 minutes.

4. Mix with 50 µL Buccutite™ FOL-Activated HRP (Component A).

5. Incubate at room temperature for 60 minutes.

Upon receiving the kit, it should be stored at 4°C. Proper storage will ensure the kit remains stable for up to six months. Alternatively, Components A and B can be stored at -20°C. However, do not freeze the Reaction Buffer (Component C). Before opening the vials, it is recommended to warm all the components and briefly centrifuge them. Then, immediately prepare the required solutions before beginning the conjugation process. The following SOP provides an example for labeling goat anti-mouse IgG antibodies.

1. To label 25 µg of antibody (assuming the target antibody concentration is 1 mg/mL), mix 1.25 µL (5% of the total reaction volume) of Reaction Buffer (Component C) with 25 µL of the target antibody solution.

   Note: If you have a different concentration, adjust the antibody volume accordingly to make ~25 µg antibody available for your labeling reaction.

   Note: The antibody should be dissolved in 1X phosphate-buffered saline (PBS), pH 7.2-7.4; If the antibody is dissolved in glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, or use ReadiUse™ 10KD Spin Filter (Cat. # 60502 from AAT Bioquest) to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for antibody precipitation.

   Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.

   Note: The antibody –Buccutite™ MTA reaction efficiency is significantly reduced if the antibody concentration is less than 1 mg/mL.

1. Add 10 µL DMSO (not provided in the kit) into the vial of Buccutite ™ MTA.

2. Add 2.5 µL of Buccutite ™ MTA (Component B) to the working antibody solution, and mix thoroughly by pipetting several times or vortexing the vial for a few seconds.

3. Keep the antibody- Buccutite ™ MTA reaction mixture at room temperature for 30 - 60 minutes.

   Note: The antibody-Buccutite™ MTA reaction mixture can be rotated or shaken for a longer time if desired.

1. Make an HRP- Buccutite™ FOL solution by adding 50 µL ddH₂O into the vial of Buccutite™ FOL-Activated HRP (Component A), mix well by repeatedly pipetting for a few times or vortex the vial for a few seconds.

2. Mix a whole vial of Buccutite™ FOL-Activated HRP solution into the antibody- Buccutite™ MTA solution, mix well, and rotate the mixture for 1 hour at room temperature.

3. The HRP-antibody conjugate is now ready to use.

   Note: For immediate use, the HRP-antibody conjugate needs to be diluted with the buffer of your choice.

   Note: For longer term storage, HRP-antibody conjugate solution need be concentrated or freeze dried.

   Note: Alternatively, add antibody-Buccutite™ MTA solution mixture to the vial of Buccutite™ FOL-Activated HRP directly.

The antibody conjugate should be stored at > 0.5 mg/mL in the presence of a carrier protein (e.g., 0.1% bovine serum albumin). For longer storage, the HRP-antibody conjugates could be lyophilized and stored at ≤ –20 °C.